Gambogic acid (GA) is known for its anti-cancer activity in a phase II clinical trial. However, the detailed molecular mechanisms of its anti-multidrug resistance remain unclear. The present was designed to study the relationship between GA and multidrug-resistant protein ATP-binding cassette transporter B1 (ABCB1). GA dose-dependently inhibited ABCB1 activity levels in the in vitro Pgp-Glo assay system and increased the cellular accumulation of ABCB1 substrate adriamycin. Although GA had no significant influence on ABCB1 mRNA in the Realtime PCR assay, western blot detection indicated the compound reduced ABCB1 protein levels. Further study showed the proteasome inhibitor MG-132 reversed the GA-decreased ABCB1 level and prolonged half-life of ABCB1. It was also found that GA coordinated with other anti-cancer drugs (such as adriamycin, docetaxel, verapamil and protopanaxadiol) to enhance cellular cytotoxicity on human epithelial cancer cell lines with higher ABCB1 expression levels. These data suggest that GA functions as a non-competitive inhibitor of ABCB1 by directly inhibiting and reducing its expression levels by promoting protein degradation through post-tranlational proteasome pathway. The results of this study will aid in the understanding of the synergistic effects of combining GA with other drugs as a new anti-multidrug-resistant agent.